Jan 3, 2007

1. INTRODUCTION

Indian sub-continent is a rich source of plant and animal wealth, which is due to its varied geographical and agro-climatic regions. Besides it's varied biodiversity, it has a diverse cultural heritage too. Though at present Indian health care delivery consists of both traditional and modem systems of medicines, both organized traditional systems of medicine like Ayurveda, Siddha and Unani and unorganized systems like folk medicine have been flourishing well. Ayurveda and Siddha are of Indian origin and accounted for about 60% health care delivery in general and 75% of rural Indian population depends on these traditional systems. These two systems of medicine use plants, minerals, metals and animals as source of drugs, plants being the major source. It is estimated that roughly 1500 plant species in Ayurveda and 1200 plant species in Siddha have been used for drug preparation (Jain, 1987, Krishnakumar and Sureshkumar, 1995). In Indian folk medicine use, about 7500 plant species are recorded as medicinal plants (Anonymous, 1996). Though the Indian traditional systems of medicine are time-tested and practiced successfully from time immemorial, there is lack of standardization with regard to identity of crude drugs, methods of preparation and quality of finished products.

Textual variations exist among the innumerable literatures on traditional medicine on the constituents of a drug, methods of preparation and the names of medicinal plants. Multitude of vernacular names of medicinal plants found in the literatures pose problems in identifying the correct botanical names of medicinal plants. And, it is worst confounded with the use of different vernacular names, for the same plant, in different localities in the country. Vernacular names of some medicinal plants whose botanical identity are not known or ambiguous, find place in standard formularies and pharmacopoeia (Example: 'Avilthol’ and 'Kiliyooral' have no botanical identity (Anonymous, 1972) and for the Sanskrit name 'Punarnava' two plants Trianthema portulacostrum, and Boerhaavia repens are mentioned (Mukerji, 1953).

Standardization of herbal drugs is most desirable at this time when world- wide interest on herbal medicine has gained momentum. Besides lack of standardization, unscrupulous commercial practice of adulterating and substituting the genuine herbal drugs are posing great hurdle in popularizing the time-tested herbal-based traditional medicine. To achieve WHO's proclamation of "Health for all by 2000 AD" traditional medical systems have to be strengthened and popularized within the shortest possible time. Standardization of herbal medicine has the key to achieve this aim.

Pharmacognosy - tool for crude drug standardization

The term pharmacognosy is derived from two Greek words 'Pharmacon' meaning drug or medicine and 'gnosis' knowledge. C.A. Seydler first coined this term in his dissertation entitled 'Analecta pharmacognosia' in 1895. Pharmacognosy is closely allied to medicine, developed during early nineteenth century as a branch of Materia Medica and applied biology. It is a study of drugs having their origin in plant and animal kingdom. The subject pharmacognosy can also be expressed as an applied science that deals with biological, biochemical, therapeutic and economic features of natural drugs and their constituents. Tyler et al. (1981) defined that in a broad sense, pharmacognosy embraces knowledge of the history, distribution, cultivation, collection, selection, preparation, commerce, identification, evaluation, preservation and use of drugs and economic substances that affects the health of men and other animals.

In the earlier days, only the external morphological characters were used to identify a drug. As late as the beginning of the present century, pharmacognosy had developed mainly on the botanical side, being particularly concerned with the description and identification of drugs both in their whole state and in powder form. Modem aspects of pharmacognosy include not only the crude drugs but also their natural constituents and their derivatives.

Like other biological sciences, pharmacognosy has utilized related fields to bridge the transition from a descriptive science to a functional science. Various pharmacognostical methods are evolved to standardize crude drugs. Therapeutic efficacy of medicinal plants depends upon the quality and quantity of chemical constituents. It has been established that chemical constituents of a plant species vary With regard to climate and seasons (Tyler et al., 1981). A plant species grown in different geographical localities also show quantitative variation in their chemical constituents (Mallavarapuet al., 1995). Variation in biological compounds exists not only in species level but also in variety and cultivars levels too. Many varieties within a species might show variations in histological and phytochemical aspects. These differences exist among varieties of commonly occurred medicinal plants. These variations might be climatic, altitudinal, geographical or genetical in nature. Many varieties of medicinal plant species are found in nature. Though pharmacognostical studies on individual medicinal plants, their constituents and their efficacious have been undertaken, little work has been done on comparative analysis of the variations in morphological, phytochemical and pharmacological aspects of varieties of medicinal plants. And to fulfill this gap, the present work is undertaken with a view to analyze, similarities and dissimilarities in morphological, anatomical, microscopical, physicochemical and phytochemical characters of the following varieties of plants. These plants are commonly available and medicinally useful in this geographical area and this study would form a foundation for understanding the pharmacological and therapeutical effectiveness of these varieties.

As stated by Metcalfe and Chalk (1957), microscopical methods are often necessary to establish the botanical identity of commercial samples of medicinal plants, timbers, fibers etc. and may play an important part in checking adulteration and substitution. It involves longitudinal and transverse sectional views of the parts of the drug.

Plant based crude drugs whose botanical identity is not known are identified based on their morphological and anatomical characters. Park et al. (1995) studied the market samples of ‘Man Byung Cho'. Based on morphological and anatomical characters of leaf midrib and leaf lamina, he concluded that they belong to the leaves of Rhododendron brachycarpum and R. brachycarpum var. roseum. Yamaji et al., (1993) after studying the anatomical characters of flower stalk and xylem vessels of rhizome established that the drug 'Spang-RtziDo-Do' is evolved from Pterocarpus hookeri. Mehrotra and Sharma (1984) analyze the various market samples of the Ayurvedic drug 'Sappan' and compare with its genuine drug Caesalpinia sappan. Using morphological and anatomical parameters, they establish the genuineness of the drug.

Organoleptical characters play an important role in the identification of crude drugs. In this method, the color, fractures, taste and smell of the drugs are characterized. Chakraborti et al. (1988) studied the stem barks of Strychnos nux-vomica and S. potatorum, and distinguished the authenticity of 'Nux-vomica' bark from other barks. Shah and Khanna (1961) distinguish the fruits of Embelia ribes with its grayish black color and warty surfaces with that of E. robusta, which has reddish, longitudinally wrinkled surface and more prominent calyx with five sepals.

Powder microscopy is another parameter used to identify and distinguish the drug from its substitutes and adulterants. For example, Patel and Satakopan (1979) distinguish Saraca asoka bark from its adulterants by the analysis of the powder and put fourth a key for the identification of the 'Asoka' bark powder. Srivastava and Srivastava (1988) identified the adulterants of Catharanthus roseus, by the analysis of powdered drug.

Physicochemical and phytochemical studies include ash value, solubility and extractive values and qualitative and quantitative analysis of phytochemicals. Satakopan and Thomas (1970) distinguish the leaves of Adhatoda vasica and its adulterant Ailanthus excelsa based on the palisade ratio and anatomical characters of leaf and petiole.

Mucuna cochinchinensis is the adulterant of the Unani drug 'Karanj' (Genuine: Pongamia Pinnata). Hasmi and Singh (1997) established it with the use of fluorescence analysis and other pharmacognostical parameters.

Quantitative estimation of major phytochemical constituents of a drug is another parameter. Liu et al. (1993) estimate the alkaloid content of three species of Phellodendron and distinguish them with one another based on quantity of alkaloid and texture and color of the herbs.

Geographical and climatic factors influence the percentage of active constituent of a medicinal plant. Dadun et al. (1992) concluded that the percentage of alkaloids content of Ephedra sinica is dependent upon the season and geographical locality in which the plant is grown. Dragur and Menary (1992) proved that the oil yield of Olearia phlogopappa is higher in summer months than in autumn. Mallavarupu et al., 1995, estimate the essential oil content of Cinnamomum zeylanicum grown in different localities. It was found that the plants grown in Hyderabad are having more oil percentage than that in the plants grown in Bangalore.

Asif and Shafiullah (1993) analyzed 175 herbal drugs with Infrared spectrum and evolved a method for checking the purity of herbal drugs. Mitra et al. (1976) after careful pharmacognostical study stated that no difference was found in the macro and micro characters in rhizome of red and white varieties of Nelumbo nucifera. Abdurahman et al. (1996) tried to distinguish the two varieties of lrvingia gabonensis based on their pharmacognostical characters.

Scanning electron microscopical (SEM) studies are also useful in the identification of crude drugs even at the variety level. Mehrotra and Shome (1993) were able to differentiate the red flowered variety with the white flowered variety of N. nucifera, by SEM studies of petals.

DNA fingerprinting is a latest tool to identify the crude drugs. Cao et al. (1996) prove that the drug 'Ku-Di-Dan' sold in Taiwan, Hongkong and Macan markets is derived from Elephantopus scaber L. With the use of artibitry primed polymerase chain reaction and random amplified polymorphic DNA he is able to pinpoint its botanical identity.

2.2. Cissus quadrangularis L.

Morphological features of C. quadrangularis, specimen collected during floristic surveys in various geographical areas have been described (Gamble, 1967, Hooker, 1978, Gileslal and Livingstone, 1978, Matthew, 1983 and Nair and Henry, 1983). Kirtikar and Basu (1980) also describe the species along with its medicinal uses. Though, in all the above works, most of the morphological characters described are in consonance, discrepancy occurs with regard to nature of tendril and season of flowering. Matthew (1983) observed that tendrils are forked or not and stout and flowers are seen throughout the year. Kirtikar and Basu (1980) described the tendrils long and slender. Flowering season of C. quadrangularis, was observed as July - December (Gileslal and Livingstone, 1978).

Metcalfe and Chalk (1957) record anatomical characters of species belonging to Vitaceae. Presence of pearl glands, mucilage cells and anomalous secondary thickenings are observed. Madan and Nair (1959) described the anatomical characters of stem of C. quadrangularis. Janardhanan et al. (1981) studied epidermal and stomatal cells of C. quadrangularis. They observed the occurrence of pearl glands on vigorously growing young stem, leaves and tendrils.

It is to be noted that in all the botanical studies on C. quadrangularis, occurrence of varieties or variants has not been recorded. However, Kumbhojkar et al. (1991) in their ethnobotanical work has noted the occurrence of round-stemmed and flat-stemmed cultivars of C. quadrangularis. A specimen of C. quadrangularis, with wingless stem (round-stemmed) has been collected by Srinivasan and deposited at Botanical Survey of India (BSI), Coimbatore (MH NO.63656 and 68684). .

Warrier et al. (1994) noted that two-sided variety of C. quadrangularis, is found in gardens. Singh and Arora (1978) mentioned that 'Birandai' is edible and 'Marol' is non-edible. Stem, stem juice, young shoots and total ash from young shoots C. quadrangularis, are medicinally useful (Chopra et al. 1956, Kirtikar and Basu, 1980 and Anonymous, 1976).

Sivasamy et al. (1991) studied the positive effect of mutagenic activity of the fruit of C. quadrangularis, against various strains of Salmonella typhimurium. Alcoholic extract of aerial parts of C. quadrangularis was tested for insecticidal property against Musca domestica and Tribolium castaneum, but found inactive (Atal et al., 1978). Antimolluscidal activity of leaf extract (alcoholic) was tested against Biomphalaria pfe~fferi and Bulinus truncatus but with negative results (Abdel-Aziz et al., 1990).

Vitamin C concentration of shoots and leaves of, C. quadrangularis, is quantified as 39 mg/100 gm and that of fresh juice as 471 mg/100 gm (Anonymous, 1950). Presence of steroids was identified by Sen (1964 and 1966). Triterpenoids were isolated by Bhutani et al. (1984), Pluemjai and Saifah (1986) and Gupta and Venna (1990 and 1991).

2.3. Madhuca longifolia

Morphological characters of Madhuca longifolia, val. latifolia and M. longifolia, var. longifolia are described in various Floras and treatises of medicinal plants. (Mukerji, 1953, Anonymous, 1962, Gamble, 1967, Gileslal and Livingstone, 1978, Kurup et al. 1979, Kirtikar and Basu, 1980,Matthew, 1983, Henry et al., 1987 and Jain, 1996). The two varieties show variation in the color of the bark, number of anthers and seeds. However, there is no conformity in the descriptions of these characteristic features in the various treatises. Bark of Bassia latifolia is described as gray colored and that of B. longifolia as dark yellowish gray in Gamble (1967), but as dark colored in M. latifolia and dark brown in M. longifolia in Mukerji (1953), Anonymous, (1962) and Kirtikar and Basu (1980). Number of anthers in M. latifolia is variously given, 16 anthers roughly in 3 rows (Matthew, 1983), and 20-30 in 3 series (Mukerji, 1953, and Kirtikar and Basu, 1980). In M. longifolia also the number of anthers are given as 18 in 2 series (Matthew, 1983), and 16-20 in 2 rows (Mukerji, 1953 and Kirtikar and Basu, 1980). Discrepancies also exist in the descriptions as to the number of seeds in a fruit, 2 in M. latifolia and one in M. longifolia Matthew, 1983) and 1-4 in M. latifolia and 1-2 in var. longifolia (Mukerji, 1953 and Kirtikar and Basu, 1980).

Anatomical characters of the members of the family Sapotaceae are described by Metcalfe and Chalk (1957). Bishayee and Bhattacharya (1992) studied the plants associated with Madhuca sp., in Birbhum district of West Bengal. Literature on Siddha medicine mentions 3 types of 'Illuppai' (Tamil vernacular name for Madhuca sp.) namely 'Illuppai', 'Seemaiillupai' (exotic) and 'kaattuilluppai' (wild) (Prema, 1989). Madhuca sp. is medicinally and commercially useful. The plant parts like stem bark, corolla lobes, seeds and seed oil are used in diabetes, burns, scalds, bronchitis, rheumatism, cough, piles, galactagogue skin diseases, tonsillitis, stomach-ache, aphrodisiac and respiratory diseases and have laxative, insecticidal and piscicidal properties (Mukerji, 1953, Chopra, et al., 1956, Anonymous, 1962, Kurup et al., 1979, Kirtikar and Basll, 1980, Anonymous, 1986, Murugesamudaliar, 1988, Warrier et al., 1994, Jain, 1996 and Hill, 1996). According to Tribal Co-operative Marketing Development Federation of India Limited, the production of oil from seeds of 'Mahua' is 171 MT/year in India (Josh, 1993). Hulagu, (1993) used the oil cake of Madhuca sp., as fertilizer and found that it is moderately immobilized.

Dolui (1988 a and b) found that the fat prepared from oil of Madhuca sp. is a promising suppository base in drug preparation. Alam et al. (1983) substituted the flowers of Madhuca instead of 'Dhataki' flowers in the preparation of 'Mustakarishta'. Saxena et al. (1992) identified that the oil of M indica is one of the ingredients of 'KubjaPrasavini Taila'. Uniyal (1993 a) found that 'Sthal Madhuk' derived from M indica. Uniyal (1993 b) identified that 'Madhok' is having two types, 'Mahua' is the M indica and 'Madhupuspi' is Aisandra hutyracae.

Atal et al. (1978) found that the extract of stem bark of M. longifolia have anti-insecticidal property against housefly. Oil of Madhuca sp. has anti-insecticidal activity against Callosohruchus cinensis (Chander and Ahmed, 1986 and Ali et al. 1983) andC. macultatus (Jadhav and Jadhav, 1984). The oil is also active against Meloidogyne incognita (Lanjewar and Shukla, 1986) and for common pests of various crops and ornamentals (Sounderrajan and Nimbisan, 1993). Katole et al. (1996) reported that, 'Mahua' oil is ineffective against citrus black fly nymphs when compared to monocrotophos, phosalare and neem oil.

Oil cake of Madhuca sp. are found to be highly deleterious to the nematodes like Meloidogyne incognita, Rotylenchulus reniformis and Tylenchorhynchus hrassicae (Alam et al., 1982), control the population of R. reniformis (Patel and Patel, 1992), inhibit gall formation of M. incognita (Goswami and Vijayalakshmi, 1986) inhibit Meloidogyne sp. population (Alam, 1989) and when applied with porate 10G inhibit Meloidogyne sp. population (Sundarababu and Vadivelu, 1989). Padmanaban and Daniel (1993) used the oil cake of M.indica against grubs of arecanut cockchafer and Leucopholis lepidophora and found active at 2440.81 kg/ha, but inactive for larval emergence from the egg sacs of cyst nematodes when compared to neem and saw dust (Devi and Gupta, 1995). Mahajan et al. (1994) found significant raise in organic and inorganic constituents of fresh water crab, Paratelpusa jacquemontii (Ruthdum) when exposed to defatted oil cakes of M indica.

50% alcoholic extract of stem bark of M indica reveal hypotensive activity and devoid of diuretic and anticancer property and LD 50- was 1000 mglkg i.p. in albino mice. (Bhakuni et al. 1969). Seed saponins of M. longifolia are reported to have spermicidal activity at 2.0% concentration (Shetty et al., 1976) and anti-inflammatory property (Yamahara et al., 1979). However, Banerji et al. (1979) reported that seed saponin of M. latifolia has spermicidal activity at 0.03% concentration. Seed saponion has no effect on cardiovascular activity and haemolytic activity (BaneIji et al., 1981) but have cholinergic activity (Mulky and Gandhi, 1977). Leaf saponins are found to have no spermicidal or spasmolytic activity (BaneIji, et al., 1985). Pyne et al. (1981) reported that the seed cake of M. indica had no ill effect in the health of milk murrah buffaloes when added up to 15% in their diet, but Cherian et al. (1996) found that it is unpalatable to rats. Varma and Singh (1978) suggested to use desaponified seed cake as cattle feed.

2.4. Basellaalba

Morphological characters of Basella alba L. (Syn. B. rubra) are described by Anonymous (1948), Sharma (1961) Gamble (1967), Kirtikar and Basu (1980), Hooker (1978), Mattllew (1983) and Warrier et al. (1994). All these treatises, B. alba and B. rubra are treated as synonyms and describe the color of the flowers as white or red. Henry et al. (1987) treated them as same species but of different varieties. According to them the green variety is B. alba var. alba and purple variety is B. alba var. rubra.

Anatomical characters of the family Basellaceae are described by Metcalfe and Chalk (1957) and that of B. rubra by Sharma (1961). Murugesamudaliar (1988) and Prema (1989) mention the presence of two types of 'Kodippasali' - white colored vine and red colored vine.

Alam (1981) reported that B. alba is a host of the root-knot nematode Meloidogyne incognita. Suriachandraselvan and Narayanasamy, (1988) have successfully controlled the potato virus 'Y' infection in chilli with the leaf extract of B. rubra.

Atal et al., (1978) found that the alcoholic extract of B. rubra have anti-insecticidal activity against Musca domestica and Tribolium casteneum.

3.1. Collection of plant materials

Plant parts of Madhuca longifolia were collected from 25-30 year old trees, from a temple-owned grove, in a village, Rajendrum Arcot of Thanjavur district, Tamilnadu. Basella alba and Cissus quadrangularis are collected from the plants maintained at HerbalGarden, TamilUniversity, Thanjavur.

Collected plant specimens were identified with the use of local Floras viz. Gamble (1967) and Matthew (1983). Identity of plant specimens was confirmed in consultation with Botanical Survey of India (BSI) Coimbatore. Identity of Variant II of C. quadrangularis was confirmed by the use of herbarium sheets available at BSI, Coimbatore (Sheets No: MH No. 63656 and ME No. 68684).

3.2. Anatomical methods

Collected plant parts were fixed in FAA (95% Ethyl alcohol 50 parts + Glacial acetic acid 5 parts + Formalian 10 parts + Distilled water 35 parts) in the field itself. Free-hand sections paraffin embedded sections and macerations were employed wherever necessary. Drawings were made with the use of prism type camera Lucida and microphotographs were taken using Leitz-Orthoplan microscope. Calcium oxalates were photographed with polarized light in the same microscope. Micro-measurements were made by calibrating a stage micrometer with ocular micrometer.

S1. No

Distilled Water

ml

TBA ml

Ethyl Alcohol 95%

Ml

Ethyl Alcohol

100% ml

Times in hours

1

50

10

40

--

2

2

30

20

50

--

12

3

15

35

50

--

1

4

--

55

45

--

1

5

--

75

--

25

1

6

--

100

--

--

1

7

--

100

--

--

12

8

--

100

--

--

1

Dehydrated materials were infiltrated at 55-60° C and embedded in paraffin wax of melting point 56-58° C. Transverse and longitudinal sections were cut at 5-15 1.1 thickness using a rotary microtome. Haupt's adhesive with formalin (4%) was used for affixing the paraffin sections on the slides.

Staining:

The staining schedules followed were,

Toluidine Blue '0'

Safranin 'a' and Fast Green 'FCF'

Toluidine Blue '0':

Differential staining of sections was achieved without removing the paraffin by using 0.05% toluidine blue in water. After staining and washing in distilled water, the slides were dried, de-waxed in xylene and mounted in DPX.

Safranin '0' and Fast Green 'FCF'

2.25 gm of safranin was dissolved in 225 ml of 95% alcohol. One gm of fast green was dissolved in a mixture of clove oil and absolute alcohol (in the ratio of 75:25). The de-waxed slides were stained in safranin for sufficient time and washed in alcohol until the excess stain was removed. Then the slides were stained in fast green and differentiated in clove oil and alcohol. The slides were brought down to xylene and mounted in DPX.

Histochemical stains

Phloroglucinol

This stain was used to localize lignified cells. Sectioned plant parts were flooded in 1 % solution of phloroglucinol in ethanol for 1-2 minutes. Excess stain was drained off. To this few drops of HCI was added. It was added and excess HCI was drained off. It was mounted in week glycerin and observed.

Ferric chloride

Localization of tannin was achieved by use of ferric chloride. Sectioned materials were kept in 10% formalin containing 2% ferric chloride. Stained sections were washed and mounted in water.

Iodine - Potassium Iodide

It was used to stain starch grains in the cells. 2 gm of potassium iodide and 0.2 gm of iodine was dissolved in 100 ml of water and used as reagent. Sections of plant materials were mounted in the above reagent and observed.

Sudan black 'B'

Sudan black 'B' was used to identify oil content in the cells. Sections of plant materials were soaked in 50% ethanol and then in Sudan black 'B' dissolved in 70% ethanol. The sections were differentiated in 50% ethanol for one minute and observed.

Maceration Technique

Jeffrey's maceration method was adopted. Small pieces of specimens not more than one nun in thickness were kept in a solution of equal parts of 10% chromic acid and 10% nitric acid for overnight or until the materials became' mushy' in texture. After maceration, the tissues were washed in water and stained with safranin. The macerated and stained tissues were spread on slides and mounted in glycerin for observation.

3.3. Quantitative microscopy

Peelings of upper and lower leaf epidermal layers were used for calculating the stomatal index. Leaf segments of 2 X 10 mm in size were soaked in sodium hypochlorite solution, until they got bleached. These bleached materials were used for calculating the palisade ratio, vein islet number and vein termination number. Calcium oxalate crystals were also observed in these materials and measured.

3.4. Preparation of powder

The necessary plant parts were collected and dried under shade. These dried materials were mechanically powdered after keeping them in an oven at 35°C for 24 hours. These powdered materials were used for further physicochemical, phytochemical and fluorescent analysis.

3.5. Analytical Methods

The procedures recommended in Indian Pharmacopoeia (1966) were followed for calculating total ash, acid-insoluble ash, loss on drying at 110°C and alcohol and water-soluble extractive percentages. The percentage of extractive value in different solvents was calculated by successive extraction of the sample in a soxhlet extractor with petroleum ether (60-80°C), benzene, chloroform, alcohol and water.

3.5.1. Total Ash

5 gm of plant powder was ignited in an electric furnace at 600°C in silica crucible until the sample reach a constant weight.

3.5.2. Acid - Insoluble Ash Value

Total ash obtained was heated with addition of 25 ml of dilute HCl for 10 minutes. It was filtered in an ash-less filter paper (Whatman No. 41) and the residue was ignited in the furnace to get a constant weight.

3.5.3. Loss on drying

Freshly collected and pre-weighed samples were dried in an electric oven at 110°C until reaching a constant weight.

3.5.4. Solubility percentage

Alcohol

5 gm of powdered material along with 100 ml of alcohol are shaken well occasionally for the first 6 hours and kept undisturbed for 18 hours. The liquefied extract thus obtained was concentrated in an vacuum oven and the percentage was calculated with the weight of the drug powder taken.

Water

The procedure adopted for solubility percentage of alcohol is used with chlorofonn water instead of alcohol to get the water solubility percentage.

3.6. Powder Analysis

Fluorescent analysis was carried out by using the method of Chase and Pratt, 1949. Behavior of powdered plant materials with different chemical reagents was carried out as mentioned by Kay (1938) and Johansen (1940).

3.7. Qualitative phytochemical studies

Qualitative phytochemical analyses were done using the procedures of Kokate (1994). Alkaloids, carbohydrates, tannins and phenols, fixed oils and fats, saponins and gums and mucilage’s were qualitatively analyzed. Shinoda's test was followed to analyze flavonoids.

3.7.1. Alkaloids

The extracts were dissolved in diluted sulphuric acid and filtered. The filtrate was treated with Mayer's, Dragendorfrs, Hager's and Wagner's reagents separately. Appearance of cream, orange brown, yellow and reddish brown precipitates in response to the above reagents respectively indicate the presence of alkaloids.

3.7.2. Carbohydrates

300 gm of aqueous and alcoholic extracts were dissolved in water and filtered. The filtrate was treated with concentrated sulphuric acid and then with Molisch's reagent. Appearance of pink to violet color indicates the presence of carbohydrates.

The filtrate was boiled with Fehling's and with Benedict's solutions. Fonnation of brick red precipitate in Fehling's and Benedict's solutions is the positive result for reducing sugars and non-reducing sugars respectively.

3.7. 3. Tannins and phenols

Small quantity of alcoholic and aqueous extracts were dissolved in water and to that ferric chloride solution (5%) or gelatin solution (1%) or lead acetate solution (10%) was added. Appearance of blue color with ferric chloride or precipitation with other reagents indicates the presence of tannins and phenols.

3.7.4. Flavonoids

The extract with few ml of alcohol was heated with magnesium and then concentrated hydrochloric acid was added under cooling. Appearance of pink color indicates the presence of flavonoids.

3.7.5. Gums and Mucilage’s

About 10 ml of extract was slowly added to 25 ml of absolute alcohol with constant stirring. Precipitation indicates the presence of gums and mucilage’s.

3.7.6. Fixed oils and fats

A drop of concentrated extract was pressed in-between two filter papers and kept undisturbed. Oil stains on the paper indicate the presence of oils and fats.

3.7.7. Saponins

About one ml of the alcoholic and aqueous extracts were dissolved separately in 20 ml of water and shacked in a graduated cylinder for 15 minutes. Formation of one cm layer offoam indicates the presence of saponins.

3.8. Quantitative phytochemical tests

3.8.1. Estimation of Ascorbic acid or Vitamin 'C'

Ascorbic acid or vitamin 'C' is estimated following the procedure in AOAC (Anonymous, 1980).

Bromine water: Few drops of liquid bromine was dissolved in water under cooling.

Standard solution: 100 mg of ascorbic acid was dissolved in 100 ml of 4% oxalic acid in a standard flask. Working standard was prepared by dissolving 10 ml of standard solution with 90 ml of 4% oxalic acid. The concentration was 100 mg/ml. It was converted to dehydro- form by adding bromine water. When it turns orange in color, it was blown with air and the excess of bromine was removed.

Standard Curve

Different aliquots (0.2 to 2 ml) of dehydro- form of working standard was taken in test tubes and their volume was made to 3 ml with water. To each tube was added 1 ml of DNPH and 1 or 2 drops of thio urea. The tubes were incubated at 37°C for 3 hours. After incubation the orange-red oxazone crystals formed was dissolved by adding 7 ml of 80% sulphuric acid. The absorbance was measured at 540 urn and a standard grape was plotted.

Sample preparation

0.5 gm of sample was ground well in a pestle and mortar with oxalic acid and filtered. The filtrate was made-up to known volume with oxalic acid. Known volume of (10 ml) the above was changed to dehydro form using the procedure adopted for working standard.

Estimation in sample

Dehydro-form of sample was taken in aliquots and preceded as for plotting the standard curve. The absorbance was compared with the standard grape and the percentage of ascorbic acid was calculated.

3.8.2. Estimation of tannins

Estimation of tannins was carried out by using Folin-Denis reagent (Anonymous,

1980).

Reagents

Folin-Denis reagent: To 750 ml of water 100 gm of sodium tungstate, 20 gm of phosphomolybdic acid and 50 ml of 85% phosphoric acid were added. The whole mixture was refluxed for 2 hours. It was cooled and diluted to 1000 mI.

Saturated sodium carbonate solution: 35 gm of anhydrous sodium carbonate was dissolved in 100 ml of water at 70-80°C and cooled for overnight. Clear liquid was decanted and used.

Standard solution: 100 mg of tannic acid was dissolved in 1000 ml of water. Fresh solutions were prepared for each test.

Preparation of sample

5 gm of sample was boiled with 400 ml of water for 30 minutes. The extract was cooled and transferred to 500 ml flask and made-up the volume.

Preparation of standard curve

10 ml of standard solution was made up to 100 ml with distilled water. 1-10 ml aliquots were taken in clear test tubes. 0.5 ml of Folin-Denis reagent and one ml of sodium carbonate solution were added to each tube. Each tube was leveled to 10 ml with distilled water. All the reagents in each tube were mixed well, kept undisturbed for about 30 minutes and read at 760 nm against reagent blank.

Estimation in sample

An aliquot of the sample extract containing not more than 0.1 mg of tannic acid was used and the percentage of tannin was determined.

Reagents

Standard solution: 100 mg of catechol in 100 mI of water. It was diluted to 10 times for working standard.

Preparation of standard curve

Different aliquots of working standard were taken in test tubes and their volume was made up to 3 mI with water. 0.5 mI of Folin-Ciocalteau reagent was added to each tube and kept for 3 minutes. To each tube, 2 ml of 20% sodium carbonate solution was added and mixed thoroughly. The test tubes were kept in boiling water exactly for one minute and cooled down. The absorbance was measured at 650 nm against blank and a standard grape was plotted.

Preparation of sample

One gm of sample was ground in a pestle and mortar with 80% alcohol. Alcoholic extract was centrifuged at 10000 RPM for 20 minutes. The supernatant was saved separately and the residue was re-extracted with 80% alcohol and centrifuged for more than five times. The supernatants collected was evaporated to dryness and dissolved in known volume of water.

Estimation in sample

Aliquots of sample were taken in test tubes diluted with water and absorbance read as above and using the standard grape concentration of phenol (as mg/100 gm sample) was calculated by using the standard graph.

3.8.4. Estimation of Sucrose As Invert Sugar

Estimation of sucrose as invert sugar was carried out by using modified Fehling's solution (Soxhlet) (Anonymous, 1980).

Reagents

Copper sulphate solution: 34.639 gm of copper sulphate was dissolved in water and diluted to 500 ml.

Alkaline tartrate solution: 17 3 gm of potassium sodium tartrate and 50 gm of sodium hydroxide were dissolved in water and diluted to 500 mi. Let it stand for two days and the clear solution was decanted and used.

2) Standard solution: 9.5 gm of pure sucrose with 5 mI of HCI was dissolved in water and made up to 100 mi. It was stored for three days at 20-25OC and then diluted to 1000 ml. Necessary quantity can be neutralised with I N NaOH before use.

Standardization of standard solution

10 or 25 ml of mixed Soxhlet solution was pipette out in a conical flask. To this was added neutralized and diluted standard solution as to reduce the copper content of reagent. It was boiled on a wire-gauge at moderate boiling for 2 minutes. One ml of (0.02%) methylene blue was added and ended the titration by adding few drops of sugar solution to get faintest blue precipitate.

Preparation of sample and estimation

The powder was boiled in alcohol, filtered and made up to 100 ml. The volume (ml) of Soxhlet reagent required to reduce all the copper content as per the procedure for standardization was calculated.

Calculations

mg of sugar in 100 ml of sample

=

Total reducing sugar required X 100

_______________________________

Titer value

3.8.5. Estimation of Total Saponins

Saponins were quantitatively estimated by the procedure followed by Bamuji et al.

(1985) with some modifications.

Estimation

Known quantity of the sample powder was extracted with alcohol in a Soxhlet extractor. The extract was concentrated in vacuum and re-extracted with petroleum ether, ether and ethyl acetate sequentially. The defatted residue was dissolved in least amount of ethanol and poured in drops in excess acetone with constant stirring. The precipitate was filtered and re-extracted for many times to get the purified saponins in acetone. The final precipitate was filtered in a pre-weighed filter paper and the percentage was calculated with the weight of the sample taken.

3.9 TLC studies

Alcoholic extracts were spotted on Silica Gel' G' coated plates. N-butanol, glacial acetic acid and water in the ratio of 4: 1:2 are used as mobile phase for identification of anthocyanins (Wagner et al., 1984).

3.10. Standardization of oil from seed kernels

Best solvent for extracting more percentage of oil from kernels was determined, by extracting the coarsely powdered seed kernels in a Soxhlet extractor with different solvents like hexane, petroleum ether, and chloroform.

Refractive index was found out with an Abbe's type refractometer and specific gravity was calculated with a standard pycnometer. Saponificaion value and iodine value are calculated by the standard procedures (Anonymous, 1980).

3.10.1. Saponificaion value

One gm of oil was dissolved in 10 ml of ether/ethanol (2: 1 v/v) mixture. To this, 25 ml of 0.5 N alcoholic potassium iodide was added and boiled in water bath for 30 minutes, with an air condenser, which was used to avoid loss by evaporation. After boiling, it was cooled and titrated against 0.5 N HCl, using phenolphthalein indicator. Simultaneously another flask was processed with out oil, as blank.

Hanus iodine solution: 13.2 gm of pure iodine was dissolved in 1000 ml of glacial acetic acid and to that 3 ml of bromine water was added.

Determination

One gm of oil was dissolved in .10 ml of chloroform. To that, 25 ml of Hanus iodine solution was added and kept in dark for 30 minutes with occasional shaking. After 30 minutes, 10 ml of 15% potassium iodide and 100 ml of freshly boiled and cooled water was added and mixed well. It was titrated against 0.1 N sodium thio sulphate, until the solution turns colorless. A drop of starch solution was added and titrated again, until it turns colorless. Simultaneously, an empty flask without oil is also processed, as blank.